Hydrazine derivatized cells

ABSTRACT

A method is provided for the preparation of semisolid particles or cells that have been chemically derivatized resulting in hydrazide functionalities covalently incorporated onto cell surface membrane molecules of the semisolid particles or cells, as well as a method for determining the levels of hydrazide groups on the particles or cells. The method involves the preparation and analysis of red blood cells chemically derivatized in such a manner that hydrazide functionalities have been covalently incorporated onto the cell membrane molecules, the preparation of hydrazine derivatized cells and the use thereof of the novel cells in agglutination reactions to promote the attachment of antigens and antibodies to the hydrazine derivatized cells without loss of antibody activity or the blocking of the etitope of interest.

This is a division of U.S. patent application Ser. No. 08/113,145 filedAug. 27, 1993, now abandoned.

FIELD OF THE INVENTION

The present invention relates to the preparation and analysis of cellsthat have been chemically derivatized in such a manner that hydrazidefunctionalities have been covalently incorporated onto the cell surfacemolecules. In yet another aspect the invention relates to a methodologyfor determining the levels of hydrazide groups on solid or semisolidparticles or cells.

BACKGROUND OF THE INVENTION

Historically, cells such as erythrocytes and their derivatives have beenutilized in diagnostic tests, such as hemagglutination, for many years.British Patent 1244 344 and also U.S. Pat. Nos. 3,714,345 and 3,715,427to Hirata disclose methods for the preparation of erythrocytesderivatized with pyruvic aldehyde and formaldehyde for use inhemagglutination reactions. Human type A erythrocytes could be coatedwith antigens such as bovine serum albumin, bacteriophages andgamma-globulins, and used in agglutination reactions. However, thecoating process does not control the orientation of coupling of theantigen.

Hydrazine-derivatized materials such as latex particles andchromatography resins, have been coupled to antigens and antibodies.U.S. Pat. No. 4,421,896 to Linneaus C. Dorman describes the preparationof hydrazine-derivatized latex particles for use in latex particleagglutination reactions. Hydrazinolysis of amide groups yieldedcarboxylic hydrazide groups after about 7 to 8 hours at a temperature of50 to about 80 degrees C. However, such conditions are much too rigorousfor cellular membranes. Additionally, cellular membranes are nottypically composed of mostly carboxylic amide groups.

Methods for immobilization of antibodies to solid supports are alsoknown. For example, antibodies can be bound to solid supports bycovalent bonds between aldehydes generated on the carbohydrate sidechains of the antibody and hydrazide groups on the solid support. Thehydrazide affinity supports the binding of immunoglobulins from avariety of species and the immobilized antibodies retain more of theirbiological activity when compared to similar pore-size supportsemploying protein non-site directed immobilization chemistry.

Jonathan M. Gershoni et al., in Analytical Biochemistry 146:59-63 (1985)disclosed the preparation of adipic dihydrazide derivatized enzymes.Gershoni et al. did not apply this method to cellular membranes, whichare much more sensitive to reaction conditions and reagents thanproteins in solution.

In view of the foregoing limitations of prior art, it is an object ofthe present invention to provide a methodology for the preparation andquantitation of semisolid particles or cells that have been chemicallyderivatized, resulting in hydrazide functionalities covalentlyincorporated onto the surface molecules of the semisolid particles orcells, as well as a method for determining the levels of hydrazidegroups that are on particles or cells.

Another object of the present invention is to prepare derivatized cellsuseful in agglutination reactions, permitting the controlled attachmentof antigens and antibodies to the cells. Hydrazine derivatized cellspermit such controlled attachments. Yet another object of the inventionis to produce hydrazine derivatized cells under conditions whichmaintain the integrity of the cellular membrane.

A further object of this invention is to attach antigens and antibodiesto hydrazine derivatized cells without loss of the antibody activity orthe blocking of the antigen epitope of interest.

An additional object of the invention is to achieve optimal antibodycoupling by incorporation onto stabilized red blood cells of optimumlevels of hydrazide functionalities.

SUMMARY OF THE INVENTION

The foregoing objects and related advantages thereof are obtained by thepresent invention in providing for the preparation and use of hydrazinederivatized cells. The invention involves the preparation and analysisof, for example, red blood cells that have been chemically derivatizedin such a manner that hydrazide functionalities have been covalentlyincorporated into their surface molecules. The preparation of hydrazinederivatized cells and their use in agglutination reactions enablepreparation of a chemically activated red blood cell to which oxidizedantibodies (glycoproteins) can subsequently be covalently attachedthrough their carbohydrate residues. The hydrazine derivatized cells areproduced under conditions that maintain the integrity of the cellularmembrane while promoting the attachment of antigens and antibodies tothe hydrazine derivatized cells without loss of the antibody activity orthe blocking of the antigen epitope of interest. The hydrazide formationon a semisolid, for example, red blood cells in an aqueous media,enables mild and stable coupling of antibodies on the red blood cells.The inventive methodology provides for determining the levels ofhydrazide groups that are on solid or semi-solid particles or cellsinclusive of red blood cells.

The present invention also provides a novel technique for detectinghuman leukocyte antigen (HLA) alloantibodies. HLA antigens that havebeen extracted from transformed lymphocytes are immobilized onto fixedred blood cells through a monoclonal antibody framework (MoAb). The HLAantigens are then detected by mixing test sera with the HLA sensitizedred blood cells in an agglutination tray. After a suitable incubationperiod, agglutinated positives are visually differentiated from thenegatives.

The technology of the invention provides for a front line screeningmethodology for detecting the presence of alloantibodies in multiparousserum samples. The specificities of the positive sera are laterdetermined using complement dependent cytotoxicity (CDC). Themethodology according to the invention provides for allosera detectionby agglutination of HLA sensitized red blood cells.

The utilization of the assay described herein for the front endscreening of multiparous sera provides a number of advantages. Theseadvantages include a constant source of antigens from lymphoblast asgrown in culture, an assay that does not require complement, reagentsthat are reasonably stable, an assay configuration and format that isamenable to automation and the potential for eliminating false positivescaused by auto antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features and advantages of the present invention are set forthin the appended claims, which are preceded by a detailed description ofembodiments in accordance with the invention, with reference to thefollowing drawings.

FIG. 1 shows a graph of a log fluorescence shift obtained as a result ofthe activation of duracytes with hydrazine; the peak on the left is forduracyte, while the peak on the right is for a hydrazine derivatizedduracyte (hydracyte);

FIG. 2 is a graph showing correlation between the OD405 obtained fromthe Ellmans test (indirect measure of hydrazide formation) and the logfluorescence, LFL2, that is, a result of hydrazide formation;

FIG. 3 is a graph showing log fluorescence shift obtained from bindingof anti-mouse-PE to duracytes (left) and W6/32-hydracytes (right);

FIG. 4 is a graph showing fluorescence shifts obtained from bindinganti-mouse-PE to W6/32-hydracytes that were prepared with variousconcentrations of W6/32;

FIG. 5 is a graph showing log fluorescence shifts obtained from bindinganti-B2M to W6/32-hydrazides (left) and HLA-hydracytes (right);

FIG. 6 is a graph showing fluorescence shifts obtained from the bindingof anti-B2M-PE to various HLA-Hydracytes that were prepared withdifferent HLA and W6/32 concentrations.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

The following abbreviation definitions are applicable to the presentinvention and utilized throughout this specification:

Abbreviation: HLA, human leukocyte antigen; HAMA, human anti-mouseantibodies; ELISA, enzyme linked immunoassay; EDAC,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; MES,(2-[N-Morpholino]ethanesulfonic acid); PBS, phosphate buffered saline;DMF, Dimethylformamide; TBL, thiobutyrolactone; OD, optical density(light absorbance) at the specified wavelength; EDTA,ethylenediaminetetraacetic acid; B2M, beta-2-microglobulin; PE,phycoerythrin; FL, fluorescence; LFL, Log fluorescence; CDC, complementdependent cytotoxicity; EBV, Epstein-Bar virus; MoAb, Monoclonalantibody; CYNAP, cytotoxicity negative adsorption positive; PRA, panelreactive antibody.

The invention enables the controlled coupling of receptors or substratesto a cell membrane. The cell surface is derivatized with hydrazine,under mild conditions to avoid destruction of the membrane, to yieldhydrazide groups on the cell surface. The receptor or substrate ofinterest is derivatized with an aldehyde group at a location such thatthe binding site or epitope remains available to its binding partnerafter coupling to a cell membrane. The coupling of the receptor orsubstrate to the cell surface can then be accomplished by reaction ofthe aldehyde group on the receptor or substrate and the hydrazide on thecell surface.

The invention can be utilized with living, dead or dormant cells, suchas erythrocytes, duracytes or bacteria and the like. Stabilized cells,such as stabilized red blood cells (for example, Duracytes, as disclosedin U.S. Pat. Nos. 3,714,345 and 3,715,427, assigned to AbbottLaboratories), and those prepared according to the methodology describedin British Patent 1 244 344, are most suitable.

Hydrazide groups can be formed on a cell surface by reacting carboxylicacid groups located on the surface of cellular membranes with hydrazinein the presence of a water soluble activating agent, such as EDAC. Thereaction can be performed by simultaneous addition of the reagents.After the reaction is complete, the approximate number of availablehydrazides on a cell can be determined. The hydrazide groups are reactedwith thiobutyrolactone to form a thiol derivative. The quantity ofthiols present can be determined with a thiol detection reagent, such asEllmans Reagent [5,5'-dithio-bis(2-nitrobenzoic acid)]. The intensity ofthe color developed in the detection reagent reaction can be correlatedto the quantity of thiols present on the cell membrane, and thereby thequantity of hydrazide groups.

Proteins can be derivatized with aldehyde groups through the use of avariety of well known reagents and methods. Preferably, a carbohydrategroup located on the protein, such as glucose, is oxidized with anoxidizing agent such as sodium periodate or the like, to a dialdehydederivative. Other aldehyde forming reactions are well known to thoseskilled in the art and could easily be adapted for this use withoutundue experimentation.

This invention is particularly useful for preparation of cells coupledto antibodies. The preferred cell is a duracyte (red blood cell) whichis stable and readily detectable. The duracyte is reacted with hydrazineand EDAC to form a hydracyte. The carbohydrate groups on the Fc portionof the antibody can be oxidized to aldehydes with sodium periodate andthe like, and the hydracyte can then be coupled to the aldehyde groupsby reacting the hydracyte and the derivatized antibodies. Theantibody-coupled hydracytes are then used in agglutination reactions todetect the presence or quantity of antigen present in a sample.

The antibody-coupled hydracytes can also be used to couple an antigen tothe cell. An antibody specific to a particular epitope on an antigen iscoupled to the hydracytes and when this antibody-hydracyte is exposed tothe antigen, the antigen binds to the antibody. Thus, the cell iscoupled to the antigen through the selected epitope. The presence orquantity in a sample of an anti-antigen antibody to a secondary epitopeon the antigen can be determined from the resulting agglutination uponexposure of the sample to the antigen coupled cell. A similar result canbe obtained using antigen coupled cells obtained through direct couplingof the antigen to the hydracyte.

Other methods of detecting the presence or quantity of anantibody-antigen complex are well known and equally applicable. Forexample, homogeneous or heterogeneous immunoassays utilizing antibodiesor antigens labeled with enzymes, such as alkaline phosphatase,horseradish peroxidase and the like, and fluorescent compounds, such asfluorescein and the like, can detect antibody-antigen complexes. Oneskilled in the art could readily adapt these methods of detection to theprinciples of the present invention without undue experimentation.

The following discussion of materials and methods inclusive of examplesare illustrative of the invention and are in no way to be interpreted aslimiting the scope of the invention as defined in the claims. It will beappreciated that one skilled in the art can conceive of many othermethods of use in which the present inventive concepts can be applied.

Materials and Methods Antibody Reagents

Rabbit anti-B2M-PE, goat anti-mouse and goat anti-mouse-PE were obtainedfrom Sigma Chemical Co. (St. Louis, Mo.)

Serum Reagents

Human anti-OKT3 sera were purchased from the University of Cincinnati(Cincinnati, Ohio). Human anti-HLA allosera, whose PRA and specificitieshad been determined using the standard no-wash NIH CDC methodology, andhuman normal sera samples were purchased from the University Hospital ofLeiden, Netherlands.

Stabilized Sheep Erythrocytes

Stabilized sheep erythrocytes (Duracytes) were prepared from sheep bloodusing the methodology described in U.S. Pat. No. 3,714,345, herebyincorporated by reference. Initially, duracytes were prepared from thebleeds of single animals; subsequently, pools of blood obtained from14-20 animals were used.

EXAMPLE 1 Hydrazine Duracytes (Hydracytes)

Hydrazine derivatized duracytes were prepared using a Radiometer pH stat(ABU 91 AutoBurette) to maintain the reaction pH. Thirty-seven mL of a13.3% solution of duracytes in 0.01M potassium pyrophosphate (pH 6.0)were added to a 125 mL Radiometer reaction vessel. This suspension wasstirred using a small stir bar with the stir rate of the Radiometerstirrer set at "3". Seven and one-half mL of a 50% hydrazinemonohydrochloride aqueous solution (pH 6.0) were added to thesuspension, so that the final hydrazine solution concentration was aboutseven and one-half percent by weight. The pH was then adjusted manuallyto 4.8 with 1.2M hydrochloric acid. Five mL of an EDAC solution (0.2mg/mL in 50 mM MES, pH 6.0) were added to the suspension. The pH of thereaction was maintained at 5.0 by the automated addition of 1.0Mpotassium pyrophosphate. After approximately 20 hours, the reaction wasterminated by centrifugation of the reaction mixture. The hydrazinederivatized duracytes (hydracytes) were then washed several times withPBS. A final 10% suspension of cells was stored at 4° C. in PBScontaining 0.1% sodium azide.

In the foregoing example water soluble EDAC was used, however, otherwater soluble derivatives of EDAC can be used as well as water solublecarbodiimides, such as, for example,1-cylohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate.

EXAMPLE 2 Hydrazine Quantitation

The levels of hydrazide generated on the stabilized erythrocytes weredetermined in the following way. Two hundred uL of each hydracytesuspension were added to each of three 2.0 mL microfuge tubes and twohundred uL of underivitized duracyte (controls) were added to a secondset of 2.0 mL microfuge tubes. Eight hundred uL of saline was added toeach tube. The erythrocytes were then vortexed and centrifuged using abench top centrifuge. Following centrifugation, the supernate wascarefully removed. The erythrocytes were then washed three more timeswith 1 mL of saline. After the last centrifugation step 400 uL of 0.5Mpotassium pyrophosphate (pH 5.0) were added to the erythrocyte pelletand the erythrocytes were resuspended by vortexing fifty uL of DMF:TBL(5:2) and the solution was added to each tube. The tubes were vortexedand rotated for five hours (room temperature) on a Labquake rotator.After five hours, 800 uL of saline was added to each tube. The cellswere then centrifuged and washed five times with 1 mL of saline. Afterthe last wash, 200 uL of Ellmans reagent (4 mg/mL in 0.1M sodiumphosphate, pH 8.0) was added to each erythrocyte pellet (approximately200 uL). The erythrocytes were vortexed to completely resuspend thecells. After two minutes, the erythrocytes were centrifuged. One hundreduL of each supernate were diluted with 1.9 mL of saline. Absorbancereadings were then made at 405 nm. The levels of hydrazide formationwere obtained by subtracting the control (duracyte) reading from thehydracyte reading and using a molar extinction coefficient of 1.36×10⁴/cm-M. Since the number of erythrocytes in the pellet is approximately2.9×10⁵, an OD₄₀₅ of 0.3 (typical value) would translate to 1.87×10¹¹hydrazides per erythrocyte.

Flow cytometry was used to follow the activation process. To do this, 10uL of the reaction was removed from the reaction vessel at varioustimes, and mixed with 1.0 mL of PBS. The fluorescence of the dilutedsample was measured on an Epics Profile II Flow Cytometer (CoulterCorp., Hialeah, Fla.). Prior to measurement, standard alignment andfluorescence checks were performed. The data were first collected in atwo parameter histogram of size (FS) vs granularity (SS). Bitmaps weredrawn around the major density. Histograms of log orange fluorescence(LFL2) vs cell number and orange fluorescence (FL2) vs cell number weregenerated from the date obtained for the cells in the bitmap. Foranalysis approximately 10,000 cells were collected in the bitmaps.

EXAMPLE 3 Antibody Isolation

Hybridoma W6/32, obtained from the American Type Culture Collection(Rockville, Md.), was grown in hollow fiber devices using standardprocedures. The W6/32 monoclonal antibody was isolated from the hollowfiber fluid by performing a 50% saturated ammonium sulfate cut. Theantibody was then dialyzed extensively against PBS followed by 0.1Msodium acetate, 0.15M NaCl, pH 5.0. The antibody solution was thencentrifuged for 1 hour at 35,000 rpm at 10° C. in a Beckman L7-65ultracentrifuge (Ti-45 rotor) for one hour to remove high molecularweight aggregates. The protein concentration was then determined usingan absorption coefficient of 1.4 for a 1 mg/mL solution when measuringat 280 nm. In order to ensure that the absorbance measurement was notinfluenced by light scattering of aggregates, a scan between 320 nm and245 nm was run. If the OD320/OD280 was greater than 0.1 and/or the OD₂₈₀/OD₂₅₀ was less than 1.9, the protein solution was reclarified byultracentrifugation.

EXAMPLE 4 Antibody Oxidation

The antibody was oxidized with sodium metaperiodate following theprocedure described by Hoffman and O'Shanessy (Journal of ImmunologicalMethods, 112, 113 (1988), hereby incorporated by reference. For each mLof protein to be oxidized (at 6.0 mg/mL) 50 uL of a sodium metaperiodatesolution (100 mg/mL in H20) were added. The reaction vessel was invertedby hand to mix the reagents and then rotated gently for 2 hours between4° C. and 8° C. After 2 hours, the reaction was quenched by the additionof a glycerol solution (100 uL of a 0.2M solution in H20 per mL ofprotein solution). The quenched solution was then dialyzed against theacetate-NaCl buffer and reclarified by ultracentrifugation. The extentof oxidation was determined using the method described by Ahn, et al.,"Use of Fluorescein Hydrazide and Fluorescein Thiosemicarbazide Reagentsfor the Fluorometric Determination of Protein Carbonyl Groups and forthe Detection of Oxidized Protein on Polyacrylamide Gels", Anal.Biochem., 161, 245 (1987), hereby incorporated by reference. Under theseoxidation conditions, approximately 3.6 aldehydes were generated foreach antibody molecule.

EXAMPLE 5 W6/32-Hydracytes

Oxidized W6/32 was coupled to hydracytes in the following manner. Two mLof a 10% suspension of hydracytes were placed in a 2.0 mL Sarstedtplastic vial. The suspension was centrifuged and the supernate wasremoved leaving behind 200 uL of packed hydracytes. Eight hundred uL ofoxidized antibody was added to the vial. The packed hydracytes wereresuspended in the solution and then the reaction was rotated for 24hours at room temperature using a Labquake Shaker (Labindustries, Inc.).After 24 hours, the reaction mixture was centrifuged and the supernatewas removed. The cells were washed twice with PBS, coated with a 1.0%solution of human serum albumin (pH 8) for 90 minutes at roomtemperature, rewashed with PBS, and then resuspended to the originalvolume with PBS-azide. The suspensions were stored at 4° C.

Transformed Lymphoblasts

Blood from selected human donors was drawn at the Blood Center ofSoutheastern Wisconsin (Milwaukee, Wis.) and shipped to the CoriellInstitute (Camden, N.J.) where lymphocytes were isolated and Blymphocytes transformed with EBV. Transformed lymphoblasts were returnedto Abbott Laboratories where they were grown in liquid suspensionculture. The HLA Class I phenotypes of the lymphocytes were obtained atthe Blood Center of Southeastern Wisconsin, before and aftertransformation, using the NIH microlymphocytotoxicity assay.

EXAMPLE 6 Solubilized HLA

HLA was solubilized, using minor variations of the procedures describedby Parham in "Purification of Immunologically Active HLA-A and -BAntigens by a Series of Monoclonal Antibody Columns", J. Biochem., 54,8709 (1979), hereby incorporated by reference, from transformed cellsthat were grown in culture. Briefly, for each gram of frozen cell pellet1.34 mL of 10 mM Tris, 0.025 sodium azide, pH 8.0 were added, followedby 0.71 mL of a 20% Brij solution (13.3% Brij 99, 6.7% Brij 97) and 0.16mL of a 20 mM iodoacetamide solution (in 10 mM Tris-azide, pH 8.0). Theresulting suspension was stirred for 2 hours at 2° to 8° C., transferredto 2.0 mL microfuge tubes and then centrifuged for 5 minutes on a benchtop centrifuge. The clear supernate was removed and transferred to a newcentrifuge tube. Care was taken to avoid transferring any floatingmaterial. The sample was centrifuged and supernate was then transferredto new tubes and stored frozen at -70° C.

EXAMPLE 7 HLA-Hydracytes

Two mL of a 10% suspension of W6/32-hydracytes were added to a 2.0 mLSarstedt vial. After centrifugation, the supernate was removed and 1.0mL of a 1:3 solution of solubilized HLA (10 to 30 ug/mL) in PBS-azidewere added to the packed hydracytes. The cells were resuspended androtated for one hour at room temperature, as previously described. Afterone hour, the cells were centrifuged, washed several times with PBS, andresuspended to the original volume with PBS-azide. The cells, in cappedvials, were then "cured" by rotating for 72 hours at 37° C. Aftercuring, the cells were transferred; without washing, to storage at 2° to8° C.

EXAMPLE 8 Serum Adsorption

In order to eliminate interferences due to human antibodies thatcross-react with mouse immunoglobulins, all human sera samples werefirst adsorbed with a combination solid-liquid phase reagent. Toaccomplish this, 2.0 mL of a 10% suspension of W6/32-hydracytes wereadded to a 2.0 mL Sarstedt plastic vial which was then centrifuged. Thesupernate was then removed and discarded. One mL of sera was then addedto the remaining 200 uL cell pellet. A nonspecific antibody of the sameisotype as W6/32 (IgG2A) was also added (200 uL of a 6 mg/mL buffer Ksolution) as well as bovine serum albumin (100 uL of a 40 mg/mL buffer Ksolution), 0.05M sodium phosphate, 0.75 sodium chloride and 0.02M EDTAat pH 7.6. The packed cells were then resuspended in the serum diluentmixture, which was then rotated for 1 hour on a Labquake rotator. Thetubes were then centrifuged and the adsorbed, diluted sera was removedfor agglutination analysis.

EXAMPLE 9 HLA Quantitation

HLA extracted from lymphoblasts was quantitated using a standardsandwich ELISA, employing rabbit anti-human B2M as the capture antibody,W6/32 as the specific antibody and goat anti-mouse alkaline phosphataseas the secondary labeling antibody. Initially, results were reported asrelative units. Later, the commercially available Sangstat sHLA ELISAkit (Menlo Park, Calif.) was used to determine actual values. For mostof the cells extracted, between 40 and 120 ug of HLA were obtained permL of extraction solution.

EXAMPLE 10 Flow Cytometry Analysis

W6/32-hydracytes and HLA-hydracytes were routinely analyzed by flowcytometry using PE labeled goat anti-mouse and PE rabbit anti-human B₂M. To accomplish this, 25 uL of W6/32-hydracytes (10%) were diluted with100 uL of PBS in 200 uL serum tubes (in duplicate). The tubes werebriefly centrifuged and supernate was removed. At this point, 50 uL of a1/5 dilution of anti-mouse-PE, or a 1/40 dilution of anti-B2M-PE, wereadded to the cells. Dilutions of the anti-mouse-PE or anti-B2M-PE, weremade using buffer that contained approximately 20% goat serum and 10%bovine serum and 0.02% Tween 20. Diluent only was added to controltubes. Extract from the Daudi cell line was also used as a control.Cells were resuspended by vortexing and rotated for 2 hours at roomtemperature. After 2 hours, the cells were washed 2 times with 100 uL ofPBS. For flow analysis 10 uL of the cell suspensions were transferred totubes containing 1.0 mL of PBS. These diluted cells were then analyzedon a Coulter Epics Profile II flow cytometer.

EXAMPLE 11 Agglutination

Agglutinations were performed by adding 32 uL of the adsorbed dilutedsera to a "V" bottom 96-well microtiter tray. Additional buffer K (13.5uL) was added along with 4 uL of HLA-hydracytes. The reaction mixturewas then thoroughly mixed. The agglutination wells were covered toprevent evaporation and the tray allowed to stand for 3 hours. Fornegative reactions, scored as "0", all hydracytes settled to the bottomof the wells and appeared as tight buttons. For positive reactions,scored "1" to "4", the buttons were more diffuse or nonexistent.

RESULTS Hydracyte Preparations

Table I shows typical values for activation levels obtained for thepreparation of hydracytes from duracytes. Each duracyte lot representspooled sheep erythrocytes from different animal bleeds. The OD₄₀₅ valueswere obtained after reacting the duracytes (controls) and hydracyteswith TBL and Ellmans reagent, as described. With regard to activationlevels, good reproducibility was seen within and between lots ofduracytes. Routinely, hydracytes were used with activation levelsranging between 1.24×10¹¹ and 3.2×10¹¹ groups per erythrocyte (OD₄₀₅between 0.2 and 0.5). If necessary, higher levels of activation can beused; however, values approaching 6.2×10¹¹ or higher can lead toirreversible clumping of the cells. FIG. 1 shows the difference betweenthe log orange fluorescence (LFL2) for duracytes (right) and hydracytes(left). Since there is a significant shift, LFL2 was used to monitor theactivation reaction. FIG. 2 shows that a linear correlation existsbetween the optical density of activated duracytes, treated with TBL andEllmans reagent, and the LFL2's of activated duracytes that weremeasured with the flow cytometer. A correlation coefficient of 0.980 wasobtained from the data in FIG. 1.

                  TABLE I                                                         ______________________________________                                        Results from the Activation of Different Lots of Duracytes with               Hydrazine                                                                                                         Activation                                Duracyte                            Levels                                    Lot #   Cell Type OD405    Delta OD405                                                                            (X10 E-11)                                ______________________________________                                        42470-50                                                                              Duracyte  0.327    --       --                                        42470-97                                                                              Hydracyte 0 609    0.282    1.75                                      41182-49                                                                              Duracyte  0.400    --       --                                        43176-13                                                                              Hydracyte 0.743    0.343    2.13                                       39972-137                                                                            Hydracyte 0.764    0.364    2.26                                      42469-19                                                                              Duracyte  0.209    --       --                                        42469-31                                                                              Hydracyte 0.473    0.264    1.64                                      ______________________________________                                    

W6/32-Hydracytes

W6/32-hydracytes were analyzed by agglutination titers with goatanti-mouse antibody by flow cytometry. The typical value obtained foranti-mouse titers was 1/64,000 and 1/128,000. These titers correspond todetection levels of 15.6 to 7.8 ng/mL of anti-mouse, respectively.

The results of flow cytometry analysis are shown in FIGS. 3 and 4. FIG.3 shows that the binding of anti-mouse-PE to W6/32-hydracytes results ina significant fluorescence shift relative to the binding ofanti-mouse-PE to control hydracytes. With increasing concentration ofW6/32 in the coupling reaction, the resulting fluorescence shiftincreases (FIG. 4). If hydracytes with activation levels lower than1.24×10¹¹ are used, lower amounts of W6/32 are coupled.

HLA-Hydracytes

The binding of HLA to W6/32-hydracytes was followed by flow cytometryusing anti-B2M-PE. FIG. 5 shows that the binding of anti-B2M-PE to anHLA-hydracyte results in a significant shift relative to thefluorescence obtained by the binding of anti-B2M-PE to W6/32-hydracytes.FIG. 6 shows that increasing the amount of HLA in the binding stepincreases the amount of HLA bound and that more HLA is bound to cellsthat have the greater amounts of W6/32 coupled to the hydracytes. It canalso be seen that at the lowest concentration of W6/32 saturation, withHLA is taking place.

Serum Adsorptions

It was found that a majority of normal and allosera react with theW6/32-hydracytes routinely included as a control. These results are notsurprising, since this type of reactivity of human serum to mouse IgGhas been extensively reported in the literature and is currentlyreferred to as HAMA (human anti-mouse antibodies). Since this reactivityis unacceptable, a few methods were evaluated to eliminate theseinterferences. It was found that the combined solid-liquid phaseadsorption procedure, was satisfactory for eliminating this HAMA. Theevaluation included 50 human sera samples from patients undergoinganti-OKT3 therapy. The HAMA ELISA tilers of these samples were between1/100 and 1/20,000. Using the solid-liquid phase adsorption, eliminationof the human anti W6/32 interference, was achieved in 49 of the 50samples. The one sample wherein the interference was not removed had thehighest ELISA titer (1/10,000) of the samples obtained.

HLA-Induced Interferences

It was found that nearly all normal sera, even after solid-liquid phaseadsorption, would show a positive agglutination when tested withHLA-hydracytes, but not with W6/32-hydracytes or W6/32-hydracytes boundwith a Daudi (control) cell extract. Daudi does not produceBeta-2-microglobulin and does not have HLA (Class I) on its surface. Theseverity of the interference correlated with the amount of HLA bound.This phenomena is herein referred to as "HLA-Induced Interference".After trying numerous methods to eliminate these interferences, it wasdiscovered that heating the HLA-hydracytes for 72 hours at 37° C. wouldeliminate these interferences. Interestingly, the amount of HLA bound tothe cells did not appear to change due to the heat "curing" treatment(as determined by anti-B2M-PE binding (data not shown)).

HLA Hydracytes

Table II shows the identity and Class I HLA specificities of the variouslymphocytes used. HLA was extracted from the corresponding lymphoblastsand then bound to W6/32-hydracytes to form the correspondingHLA-hydracytes. Also shown in Table II are the fluorescence shifts,FL2's, that were obtained when anti-B2M-PE was bound to each individualHLA-hydracyte. Since Class I HLA is composed of a unique heavy chain andcommon light chain (B2M), this measurement was used to indirectlyquantitate the total HLA bound to each hydracyte.

                  TABLE II                                                        ______________________________________                                        HLA Specificities and Anti-B2M-PE Values for Various HLA-                     Hydracytes                                                                    Cell ID #    Specificity      FL2                                             ______________________________________                                        1            A33, 34; B58, 72; Bw4, w6                                                                      351                                             2            A3, 31; B35, 60; Bw6                                                                           419                                             3            A1, 3; B35, 37; Bw4, w6                                                                        312                                             4            A2, 24; B8, 50; Bw6                                                                            446                                             5            A24, 33; B56, 65; Bw6                                                                          358                                             6            A2, 30; B55, 60; Bw6                                                                           413                                             7            A23, 36; B53, 58; Bw4                                                                          391                                             8            A11, 30; B13, 52; Bw4                                                                          429                                             9            A11, 25; B27, 44; Bw4                                                                          370                                             10           A26, -; B13, 55; Bw4, w6                                                                       404                                             11           A3, 28; B7, 63; Bw4, w6                                                                        363                                             12           A23, 24; B7, 49; Bw4, w6                                                                       360                                             13           A1, 24; B8, 48; Bw6                                                                            422                                             14           A26, 28; B8, 27; Bw4, w6                                                                       393                                             22           A2, 3; B47, 51; Bw4                                                                            346                                             28           A28, 29; B13, 35; Bw4, w6                                                                      335                                             30           A1, 33; B38, 58; Bw4                                                                           349                                             31           A23, 74; B18, 42; Bw6                                                                          405                                             502          A2, 25; B7; B38; Bw4, w6                                                                       411                                             503          A1, 32; B8, 47; Bw4, w6                                                                        377                                             504          A33, 68; B14, 48; Bw6                                                                          408                                             506          A24, 29; B7, 54, Bw6                                                                           392                                             ______________________________________                                    

Blind Agglutination Study

In order to demonstrate that the assay was capable of distinguishingallosera from normal sera, a blind study in which 163 sera obtained fromUniversity Hospital in Leiden, Netherlands, were tested. A comparison ofthe CDC results obtained at Leiden compared to agglutination resultsobtained, is shown in Table III. Fifty-eight sera were shown to bepositive in both the agglutination and CDC assays. Twenty-two sera werepositive in the agglutination assay and negative in the CDC assay (falsepositives). Nine sera were negative in the agglutination assay andpositive in the CDC assay (false negatives), and seventy-four sera werenegative in both CDC and agglutination (true negatives). From the datain Table III, a chi square value of 63.97 was obtained with a p<0.0001.These results demonstrate that the assay can be used to effectivelyidentify normal sera that are negative for HLA alloreactivity. Since ina typical sampling of multiparous sera only 10-20% of the samples wouldbe CDC positive, this test provides a convenient means for significantlyreducing the number of samples that would need to be tested by CDC.

The specificities obtained by CDC were compared with those obtained byagglutination. Of the 58 sera that were shown to be agglutinationpositive, clear specificities, by NIH CDC, were available for only 47sera. A comparison of these 47 sera is shown in Table IV. A reasonablematch was seen with 32 of the 47 sera (68%). The utilization of theassay described herein for the front end screening of multiparous serahas several advantages. These advantages include a constant source ofantigens from lymphoblasts grown in culture, an assay that does notrequire complement, reagents that are reasonably stable at 4° C. to 8°C., an assay configuration and format that is amenable to automation,and the potential for eliminating false positives due to autoantibodies.Good stability at 4° C. to 8° C. has been observed. Utilizing additivesthat would allow for freezing the reagent at -20° C. to provide extendedstability is considered possible.

Although the agglutination assay missed nine CDC positive sera, thesesera were either nonspecific (possibly due to autoantibodies) or weak inCDC. The fact that alloreactivity was detected in 22 CDC negative serasuggests that the agglutination assay might be detecting CYNAP positiveHLA alloantibodies that are otherwise missed by the standard no-wash CDCmethodology.

                  TABLE III                                                       ______________________________________                                        Concordance Results for Agglutination vs CDC                                  Results           Number of Sera                                              ______________________________________                                        True Positives (+, +)                                                                           56                                                          False Positives (+, -)                                                                          22                                                          False Negatives (-, +)                                                                           9                                                          True Negatives (-, -)                                                                           74                                                          ______________________________________                                    

                  TABLE IV                                                        ______________________________________                                        Sera ID   Agglutination                                                                              CDC       Match                                        ______________________________________                                        1708      1, 36        1+        +                                            2079      1, 36+       8         -                                            299K      2, 17        2         +                                            178G      2, 17        2, 28     +                                            97G       2, 28        2         +                                            236B      9, Bw4       1         -                                            3104      C7           25, 17    -                                            1225      NS           2, 7      -                                            168F      Bw6          Bw6       +                                            226A      2, 28, 9, 10 2, 28, 22 +                                            191B      2, 16, 17    2, 57     +                                            282C      7            7, 40     +                                            272B      NS           27        -                                            2338      NS           Cw6, Cw7  -                                            2406      9            9         +                                            174C      13           13        +                                            2259      8            8         +                                            194E      5, 35        5, 35     +                                            1236      NS           35, Bw4   -                                            2619      NS           12, 21    -                                            2002      2            2, 28     +                                            2158      35, 51, 52   5, 35     +                                            268G      2, 17        2, 28     +                                            2024      Bw4          5, 35     +                                            309C      NS           7, 40, Cw2                                                                              -                                            269H      7, 22, 40    7, 40     +                                            2215      7, 22        7         +                                            3047      NS           2, 7      -                                            2035      9, Bw4       12        -                                            2327      Multi        Bw4       -                                            237C      2            2         +                                            1348      25, 26       25, 17    +                                            147C      2+           2         +                                            1258      NS           44        -                                            3148      17           17, 11    +                                            308B      13, 15+      2, 15, 17 +                                            3137      NS           8         -                                            119B      Cw4          Cw4       +                                            2204      NS           2, 28     -                                            199J      13, 47, 7    13, 40    +                                            276F      40, 47, 7    Bw6       +                                            1247      35           35        +                                            188H      Bw6          Bw6       +                                            186F      9, Bw4       9, Bw4    +                                            618B      Bw6          Bw6       +                                            208A      7, 47, 22, 42                                                                              7, 27     +                                            2013      2, 28        2, 28     +                                            ______________________________________                                    

Numerous methods for coupling ligands to erythrocytes have been reportedin the literature. None of these methods utilizes a site-directed methodof coupling whereby the ligand (antibody) is immobilized through thecarbohydrate portion of the Fc region. It has been suggested that thissite directed coupling of antibody to solid-phase reagents is superiorto random coupling procedures due to the proper orientation of theactive site that is obtained with site-directed coupling. Site-directedcoupling of antibodies to solid phases can be achieved by oxidation ofthe antibody (to produce aldehydes) and then coupling it to the solidphase having hydrazide groups available for reaction with the aldehydegroups of the antibody. The present invention has presented methodologyto activate red blood cells (duracytes) and has shown that it can beutilized to couple active W6/32 to activated duracytes (hydracytes)which, in turn, can bind HLA and then detect HLA allosera.

Methods are presented by the invention for quantitating hydrazide groupsthat are on solid phases or cells. It has now been shown that TBL, whichreacts more rapidly and to a much greater extent with hydrazides thanwith amines at pH 5.0, can be used to convert hydrazides to sulfhydrylswhich, in turn, can be quantitated with Ellmans reagent. This providesan indirect method for quantitating the hydrazides prepared on thestabilized cells.

In addition to the Ellmans quantitation, a measurement of the cells ofthe LFL2 using a flow cytometer is a very convenient method forfollowing the actual progression of hydrazide derivatization of thecells in a stat method. This is very convenient and helpful since therate of hydrazide derivatization can vary with reaction vessel size,stir rate, temperature and pH.

While the present invention has been particularly demonstrated withreference to specific materials and examples, it will be understood bythose skilled in the art that changes in form and details can be madetherein without departing from the spirit and scope of the invention, asdefined by the following claims.

We claim:
 1. A method for the preparation of red blood cells that havebeen chemically derivatized in such a manner that hydrazidefunctionalities have been covalently incorated into the membranes ofsaid red blood cells, which method comprises chemically activating saidred blood cells and reacting said red blood cells with hydrazine to formsaid hydrazide functionalities.
 2. The method according to claim 1,wherein said chemically derivatized red blood cells are maintained in anenvironment which preserves the integrity of the membranes of said redblood cells during chemical activation of said red blood cells andreaction of said red blood cells with hydrazine.
 3. The method accordingto claim 2, wherein said red blood cells are chemically derivatized inaqueous solution and activated by an aqueous activating agent.